Targeted therapeutic nanoparticles

ABSTRACT

Disclosure of methods and compositions related to chemical conjugations to nanoparticles of polysaccharides cross-linked to poloxamers as well as nano-sized colloids comprised of polysaccharides and poloxamers. The nanoparticles may be produced by various methods including inverse miniemulsion polymerization processes which create nanogels of desired size, shape, and stability for controlled therapeutic drug delivery, imaging, and theragnostic applications.

RELATED APPLICATIONS

This patent application is a continuation of pending U.S. patentapplication Ser. No. 13/943,074 filed on Jul. 16, 2013, which claimspriority under 35 U.S.C. 119 to U.S. Provisional Patent Application No.61/672,177 entitled “Conjugation of Polymers and Hydrogels” filed onJul. 16, 2012, hereby incorporated by reference in its entirety, andunder 35 U.S.C. 120 to U.S. patent Ser. No. 13/286,320 entitled“Polymers and Hydrogels” filed on Nov. 1, 2011, hereby incorporated byreference in its entirety, which claims priority under 35 U.S.C 119 toU.S. Provisional Patent Application No. 61/344,872 filed on Nov. 1,2010, hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The invention is related to therapeutic drug delivery technologies.Polysaccharide-poly(epoxide) or more specifically,polysaccharide-poloxamer, nanogels may be functionalized with ligandsand/or targeting agents to provide a biodegradable, and thermallyresponsive drug delivery. The hydrogel nanoparticles can be“functionalized to target” specific desired body sites for thecontrolled release of encapsulated therapeutic cargo.

BACKGROUND

The use of antibiotics in the treatment of infectious disease hasincreased greatly over the past fifty years. Due to the ubiquitous use,bacteria have developed resistance to the antibiotics commonlyadministered to treat bacterial infections. Antimicrobial resistance hasbeen reported for all known antibacterial drugs currently available.

The global infectious disease treatment market is now over $100 billion,and by 2014 is expected to be $138 billion. According to the Centers forDisease Control and Prevention (CDC), each year in the US alone morethan 2 million people acquire hospital-acquired infections (HAI) ofwhich almost 100,000 die. Estimated costs associated with HAI in the USare as high as $30 billion annually. HAI and other infections are mostoften caused by gram-negative bacteria, but can also occur from otherbacteria, viruses, fungi and parasites.

The annual cost of antibiotic-resistant infections in the US healthcaresystem has recently been calculated to be in excess of $20 billion. Thewidespread use of antibiotics, especially broad-spectrum antibiotics, isbelieved to have played a significant role in the emergence of resistantbacteria.

An effective delivery system for antimicrobials would target susceptiblemicroorganisms rather than a systemic delivery to the entire body. Thisdelivery system will allow for lower levels of the agents, reduce theexposure to beneficial organisms and minimize the development ofdrug-resistant strains of pathogens. The concept of a “magic bullet”delivery system has been widely discussed in the literature but,unfortunately, few strategies have proven successful. Drug activity is aresult of molecular interaction(s) in certain cells. The drug must reachthe cellular site of action at sufficient concentrations following oral,intravenous, local, transdermal, or other means of administration. Theaim of drug delivery is to deliver the drug at the specific site ofaction, at the right concentration, and for the effective period oftime.

One method that has been employed is the use of monoclonal antibodieswith specificities directed toward specific antigenic sites on thetargets. This presumes that antigenic sites on the targets can bedefined and that for each pathogen unique antibodies can be producedthat are directed toward these sites with minimal cross-reactivity.These goals are not easily achieved. Nonetheless, a system that canoptimize cellular targeting, maintain effective intracellularantimicrobial concentrations and provide resistance to inhibit multipletargets within multiple classes of pathogens is a goal widely sought.

Gram negative bacterial pathogens constitute the most ubiquitous andserious sources of infection in civilian and military populations.Gram-negative bacteria have an outer membrane that protects them fromantibiotics and detergents. All gram negative bacteria exhibit endotoxinor lipopolysaccharide (LPS) in their outer membrane. Endotoxin iscomposed of complex carbohydrates and a lipid. The carbohydrates vary instructure and confer the antigenic properties which distinguishdifferent bacterial strains. The lipid is highly conserved acrossstrains and is responsible for many of the pathogenic effects ofendotoxin. Lipid A is the most common lipid moiety in gram negativebacteria and is a potent pyrogen and a polyclonal B lymphocyteactivator. The ubiquitous nature of endotoxin makes it an attractivetarget for treatment of nearly all gram negative bacterial strains.

There exists a need for a pharmaceutical drug delivery system that cantarget specific cells and deliver treatment to the targeted cells.

SUMMARY

In one aspect, the invention is directed to nanoparticle drug deliverysystems. The drug delivery system may comprise a nanogel targetingmolecule or nanoparticle. In one embodiment, the nanogel targetingmolecule comprises a polymeric network. The polymeric network maycomprise a plurality of first block copolymeric segments derived fromepoxide monomers and a plurality of second polymeric segments derivedfrom polysaccharides. Further the drug delivery nanoparticle maycomprise targeting agents chemically attached to the polymeric network.

In certain embodiments of the polymeric network, the block copolymer ofepoxides is a triblock copolymer. The block copolymer may comprise atleast one block derived from propylene oxide monomers and at least oneblock derived from ethylene oxide monomers. In a further embodiment, theblock copolymer of epoxides is an ABA triblock copolymer wherein the Ablock is derived from ethylene oxide monomers and the B block is derivedfrom propylene oxide monomers, such as a poloxamer. Embodiments of thenanogels and nanoparticles may be thermally responsive or degradable athuman body temperatures such as in a range of 96° F. to 100° F.

The hydrogel or hydroparticle may comprise polysaccharide monomers inaddition to the poly(epoxide) monomers. Polysaccharides are longcarbohydrate molecules of monosaccharide units joined together byglycosidic bonds that range in structure from linear to highly branched.Polysaccharides are often quite heterogeneous, containing slightmodifications of the repeating unit. Dextran is a colloidal,hydrophilic, and nontoxic polysaccharide that may be enzymaticallydegraded in the human body dextranase. Dextran is composed of linearα-1, 6-linked D-glucopyranose residues with a low fraction of −1,2, −1,3and −1,4 linked side chains. Dextrans, as well as other polysaccharides,have a plurality of hydroxyl groups that can be directly reacted to addfunctional groups to the dextran backbone or may be modified to formreactive end groups which may be used for cross-linking or otherwisefunctionalizing the hydrogel. For example, the saccharide may befunctionalized allyl isocyanate (Al), ethylamine (AE), chloroacetic acid(AC) and/or maleic anhydride (AM). Dextran fulfills many of the idealcharacteristic features of a good carrier candidate. It is nontoxic,nonimmunogenic and nonantigenic.

The nanoparticles may be functionalized with groups that are capable ofbinding with a receptor on a cell (hereinafter “targeting agents” or“ligand”) chemically attached to the nanogel or nanoparticle. Thenanogel or nanoparticle comprising chemically attached targeting agentmay then be considered a targeting molecule. In a specific embodiment,the hydrogel targeting molecule comprises polymeric segments derivedfrom dextran and polymeric segments derived poloxamers that arecross-linked and a targeting agent chemically attached to the polymericnetwork. The soft cell properties of dextran and the stabilizing,thermally responsive properties of poloxamers and poly(epoxides) offer asafe, non-toxic, and controlled drug delivery vehicle.Polysaccharide-poloxamer hydrogels, nanogels, and nanoparticles arebiodegradable, bioabsorbable and will deteriorate to elements naturallyexcreted or absorbed by the body allowing release of medicaments at thetargeted site.

Targeting agents of the nanogels or nanoparticles are capable of bindingto a receptor in the body. As used herein, a receptor is a molecule or aportion of a molecule found on the surface of a cell that receiveschemical signals from substances outside the cell. When targetingmolecule binds to a receptor, they direct the cell to do something, suchas divide, die, or allow specific substances to enter or exit the cell.Binding to the receptor may be through covalent bonding, ionic bonding,complexation, hydrogen bonding, dipole-dipole interaction, van der Waalsforces or any combination of such associations between at least one siteof the targeting agent and at least one site of the receptor, as long asthe binding is sufficiently strong to essentially form a target-receptorassociate and activate the receptor. The targeting agent may bepolymyxin B or a monoclonal, for example.

In additional embodiments, the nanoparticle drug delivery systemcomprises medicaments. The medicaments may be chemically attached to orencapsulating within the nanogel. The targeting agent attaches thenanoparticle to the target receptor and the medicaments may be releasedfrom the nanogel or nanoparticle as the poloxamer based segment isthermally or otherwise degraded and the dextran based segment isenzymatically degraded. This provides a drug delivery system withtargeted medicament delivery. The medicaments may be for therapeutictreatment of a wide variety of diseases and ailments that would benefitfrom such targeted delivery. For example, the medicament may be at leastone of a pharmaceutical, nicotinic acid, glucocorticoids and otherbudesonides, mitomycin C, monoclonal antibodies, anti-inflammatoryagents, naproxen, aspirin, ketoprofen, ibuprofen, diclofenac,indomethacin, a prodrug, a fluorescent labeling agent or radiotagbiomarker. The nanoparticle targeting nanogel or nanoparticle may havean average diameter in the range of 1 nanometer to 1000 nanometers orthe nanogel targeting molecule may have an average diameter in a rangefrom 20 nanometers to 250 nanometers, for example. An optimal size ofhydrogel nanoparticles for prolonged in vivo blood residence is in the20-200 nm range.

In another embodiment, enzymes may be medicaments chemically attached orencapsulated into the polysaccharide-poly(epoxide),polysaccharide-poloxamer or dextran-pluronic F-127 hydrogel and/ornanoparticles. The enzymes may be conjugated to dextran or poloxamersmonomers prior to polymerization or attached to the surface of thenanogel or nanoparticle. Enzymes that may be chemically attached to orencapsulated within the hydrogel include, but are not limited to,α-amylase, arginase, asparaginase, carboxypeptidase, catalase,β-galactosidase, hyaluronidase, NAD+, streptokinase, papain,α-chymotrypsin and trypsin. In other embodiments, dextran may befunctionalized to attach prodrugs by preparing carboxymethyl dextran,dextran sulphate, or diethylaminoethyl dextran. Diethylaminoethyldextran is an example of a charged dextran derivative that may formcomplexes with various chemical entities including, for example,bleomycin, isometamidium and gentamicin may form a dextran sulphatecomplexes and proteins and nucleic acids that may be chemically attachedto the hydrogel or nanoparticle. Hormones that also may be linked to thehydrogel include, but are not limited to, oxytocin and vasopressin.

Polymyxins may be conjugated to the hydrogel. Polymyxins areantibiotics, with a general structure consisting of a cyclic peptidewith a hydrophobic tail. They disrupt the structure of the bacterialcell membrane by targeting its phospholipid receptors. Polymyxins areproduced by nonribosomal peptide synthetase systems in gram-positivebacteria such as Paenibacillus polymyxa and are selectively toxic forGram-negative bacteria due to their specificity for thelipopolysaccharide molecule that exists within many gram-negative outermembranes.

Polymyxins B and E (also known as colistin) are used in the treatment ofgram-negative bacterial infections. The global problem of advancingantimicrobial resistance has led to a renewed interest in their userecently.

The invention is also directed to a method of forming a nanoparticledrug delivery system. In one embodiment, the process is directed to aminiemulsion polymerization process, such as, but not limited to, aradical polymerization miniemulsion process or a controlled radicalpolymerization process such as atom transfer radical polymerization. Inanother embodiment, polysaccharides are copolymerized with poloxamers inan inverse miniemulsion process. The polysaccharide may befunctionalized with polymerizable functional groups and/or otherfunctional groups such as medicaments or targeting agents prior to,during, or after polymerization. Similarly, the poloxamer may befunctionalized with polymerizable functional groups and/or otherfunctional groups such as medicaments or targeting agents prior to,during, or after polymerization.

Miniemulsion polymerization processes are conducted in speciallyformulated heterophase systems consisting of stable nanodropletssuspended in a continuous phase. The narrowly size distributednanodroplets of 50 to 500 nm may be prepared by a shearing systemcontaining oil, water, a surfactant, and an osmotic pressure agent whichis insoluble in the continuous phase.

Hydrogel nanoparticles are solid colloidal drug carriers, ideally in therange of 20 to 120 nm in average diameter, whereby a drug may beencapsulated within the core nanodomain and/or conjugated to thehydrogel network.

Nanoparticles comprising dextran (polysaccharides) and Pluronic® F127(poloxamers) may be produced via emulsion (colloidal) processpolymerization. For IV applications the poloxamer, Pluronic® F68, can beutilized for nanoparticle production by the same process. The smallmolecular weight poloxamer should produce nanoparticles suitable for IVadministration.

DESCRIPTION

Versatile hydrogels and nanogels comprising cross-linked polysaccharidesand epoxide polymers is described in U.S. patent application Ser. No.13/286,320 entitled “Polymers and Hydrogels” and is hereby incorporatedby reference in entirety. In one embodiment, modified dextran monomersand modified poloxamer, Pluronic® F-127, are cross-linked to form ahydrogel. This is an interesting hydrogel as both dextran and Pluronic®F-127 have been approved for use by the FDA and a hydrogel formed fromthese compounds offers a controlled drug delivery platform that isnontoxic, biodegradable, and thermally responsive at normal human bodytemperatures.

An embodiment of a polysaccharide-poloxamer hydrogel is shown inChemical Structure 1. A natural polymer, dextran is a colloidal,hydrophilic, and nontoxic polysaccharide composed of linear α-1,6-linked D-glucopyranose residues with a low fraction of −1, 2, −1, 3and −1, 4 linked side chains. Also dextran is biodegraded in the humanbody by dextranase. Dextran comprises a plurality of reactive hydroxylgroups (i.e., —OH group) that can be reacted with additional compoundsto attach functional groups to the polymer backbone. These functionalgroups may react with other monomers to form hydrogels by anypolymerization process, for example, via photochemical cross-linking.

Additionally, the dextran hydroxyl groups provide a vehicle forproducing “tunable” hydrogels. The properties of the nanogels may bevaried by varying the degree of cross-linking, the molecular weight ofthe polysaccharide and the poloxamer, the block lengths of thepoloxamer, and the addition of any other monomers, and/or chemicallinking groups, for example. Nanogels may be produced with differentmechanical properties including, but not limited to, mechanicalsolubility, electric charge, partition coefficient, strength, swellingcapacity, diffusion, thermal and/or enzymatic degradation, etc. Thephysico-chemical properties of the dextran-poloxamer hydrogel conjugatesmay be modified in order to guide the conjugate selectively to thetargeted site to increase the efficacy of the targeting agent. Thedextran-poloxamer hydrogel synthesis can be optimized per the specificapplication for controlled drug delivery and duration of prescribedtherapy, e.g., various dextran-poloxamer varying the ration ofdextran-poloxamer composition.

In one embodiment, poloxamer diacrylate may be reacted with a dextranacrylate to form the hydrogel.

Example 1: Pluronic F-127 Diacrylate Synthesis

Pluronic F-127 (poloxamer) obtained from BASF may be dissolved into a10% solution with dichloromethane (DCM) in a 2-neck flask with a stirbar. Triethylamine (TEA) and acryloyl chloride may be added to the flaskin the molar proportion of 3× excess to the [—OH] end groups of thepoloxamer and the flask stirred at 80° C. for 3 hours under reflux.After such acrylation, the solution may be filtered and precipitated inhexane to recover the poloxamer diacrylate and then dried to removeresidual hexane. This reaction may also be performed with any solublematerials that possess alcohol groups, so other polymers may also bemodified accordingly.

Example 2: Dextran Acrylate Synthesis

Commercially obtained dextran may be dissolved into a 10% solution withDMSO in a 2-neck flask with a stir bar. Triethylamine and acryloylchloride were added to the flask in the molar proportion relevant to thenumber of [—OH] end groups which are desired to be acrylated (may bevariable based upon the desired degree of cross-linking) and the flaskmay be stirred at 80° C. for 3 hours to allow sufficient conversion.After such acrylation, the dextran acrylate DMSO solution may befiltered and precipitated in isopropanol to purify the dextran-acrylateand subsequently dried. The grade and type of dextran may be chosen inorder to control the properties as well as the degree of modification.

Other methods of hydrogel formation may also be used without monomermodification steps such as a radical polymerization process. Stablenanogels of desired size and shape maybe produced with an inversemini-emulsion polymerization process such as a controlled radicalpolymerization inverse mini-emulsion process.

The polysaccharide-poloxamer hydrogel may further be conjugated withfunctional group targeting systems for site specific therapeutic drugdelivery. More specifically, polysaccharide-poloxamer nanogels (nanogelsare hydrogels which have an average diameter between 1 and 1000nanometers) may be functionalized to be biocompatible, biodegradable,bioabsorbable and thermally responsive to body temperatures that willtrigger a controlled release of medicaments.

Inverse emulsion polymerization may be used for fabrication viacross-linking of acrylate derivatives of dextran and diacrylatederivatives of poloxamers. This polymerization technique allows forcontrol over size, is versatile in respect to initiation andcomposition, and may proceed to full double-bond conversion in arelatively short time. Incorporation of functional polysaccharidecomonomers, like dextran, in the polymeric network affords thepossibility of further conjugation, such as the addition of biomarkers,fluorescent labeling, and macromolecular prodrugs, for example.Moreover, hydrogel nanoparticles in the range of 50-250 nm may bedesigned to maintain a state of stability as aqueous dispersions, resistaggregation, and can be freeze-dried as solid powders for long-termstorage without degradation.

Example 3: Dextran Modification for Further Reaction

Dextran may be functionalized directly through reaction with thehydroxyl groups or indirectly by reaction with a linking functionalgroup and subsequent further functionalization. The hydroxyl groups mayeasily react with a variety of linking functional groups including, butnot limited to, isocyanates and acyl chlorides. The dextran may beutilized with these groups or further functionalized on these linkingfunctional groups. A generalized reaction for functionalizing dextran isshown below in Reaction 1. In this case “R” and “R1” are generally anygroup that has some desirable property to be applied for this process.These modifications are as follows:

Example 4: Copolymerization with Dimethylaminoethyl Methacrylate(DMEAMA) and SiRNA

The poloxamer diacrylate (Example 1) and dextran acrylate (Example 2)monomers may be co-dissolved with DMEAMA and/or siRNA in an aqueoussolution at various concentrations prior to polymerization. Additionallyirgacure 2959 may be dissolved into this formulation at ˜1-5% w/wmonomers to make the resultant hydrogel photoactive.

Additional monomers may be substituted for or added in addition to theDMEAMA to provide additional properties to the resultant hydrogel,including, but not limited to, hydrogen bonding monomers (n-vinylpyrrolidinone, acrylic acid), thermally sensitive monomers (N-isopropylacrylamide), or additional cross-linking agents (polyethylene glycoldiacrylate) so as to add or modify properties of the resultantparticles. Examples of potential monomers are shown in Table 1.

TABLE 1 Example monomer additives Property Monomer Structure HydrophobicSolubilization/ plasticization modifiers 4- aminosalicylic acidmethylacrylate

Styrene

Stearyl methacrylate

Methyl methacrylate

Cyclohexyl methacrylate

Ethylene glycol phenyl ether methacrylate

Poly(propylene glycol methacrylate

Poly(propylene glycol) 4- nonylphenyl ethyl acrylate Hydrophiliccomponents Poly(ethylene glycol) methacrylate

Acrylamide

Hydrophilic Muco- adhesive components Acrylic acid

N- vinyl- pyrrolidinone

Thermogelling components N- isopropyl- acrylamide (NIPAM)

Vinyl- caprolactam (VCM)

2- (Diethylamino) ethyl methacrylate

Example 6: Exemplary Dextran Conjugation Chemical Processes

Direct Esterification

The dextran ester prodrugs of several drugs like nicotinic acid,naproxen, aspirin, ketoprofen, ibuprofen, diclofenac and indomethacinhave been synthesized with the aim of achieving prolonged releaseproperties. Dextran can be attached to the drug to form a prodrug by adirect linkage, attachment through linker group. In direct linkagemodel, drug is directly linked to the hydrogel, which would release theactive agent in a predictable manner by thermal degradation, pHdependent hydrolysis, or other degradation of portions of the hydrogel.

Dextran can be attached to the drug to form a prodrug by a directlinkage, attachment through linker group or through covalent bonding,ionic bonding, complexation, hydrogen bonding, dipole-dipoleinteraction, van der Waals forces or any combination of suchassociations. In direct linkage model, drug is directly linked to thehydrogel, which would release the active agent in a predictable mannerby thermal degradation, pH dependent hydrolysis, or other degradation ofportions of the hydrogel.

Carbonate Ester Method

Drugs containing a hydroxyl group can be coupled to dextran in the formof carbonate ester linkages either by activating the carrier hydroxylgroup by phosgene followed by addition of alcoholic drug (Scheme 2) orby preparing chlorocarbonate dextran esters of the drug which arefurther used as intermediates in the construction of enzyme conjugates.

Periodate Oxidation Method

Dialdehyde dextran is obtained by periodate oxidation of dextran, whichis condensed with amino compounds yielding schiff bases. The subsequentreduction with sodium borohydride is performed in order to stabilize theconjugate.

Carbamate Ester Method

The carbamate ester liganded conjugates exhibit prolonged duration ofactivity and reduced toxicity in proportion to the free drug. Theprincipal routes to obtain dextran carbamate ester linkages are shown inScheme 4.

Bromide Activation Method:

The cyanogen bromide activation of dextran is probably the most widelyused reaction to achieve covalent attachment of compounds possessing anamino function to dextran as shown in Scheme 5.

Acid Cleavable Linking Groups

Conjugates may be added to polysaccharides by acid cleavable functionalgroups such as shown in Scheme 6 to add prodrugs to the nanogel ornanoparticles.

Targeted Conjugations of Dextran

As stated above, the chemical composition of polysaccharide-poloxamerhydrogels provides for chemical modifications for attaching targetingagents for receptors and attaching or encapsulating medicaments fortherapeutic treatment of a wide variety of diseases and ailments. Dualconjugations may also be prepared, for example, apolysaccharide-poloxamer nanoparticle may comprise both a cancer cellluminescent biomarker conjugate and a cancer cell targeting prodrugconjugate for real time theranostics.

Additionally, Polymyxin B (PMB), shown in Formula 1, may be conjugatedto a polysaccharide-poloxamer nanoparticle as shown in Formula 2.Polymyxin B is an antibiotic used against gram-negative bacterialinfections. Polymyxin B is a targeting agent that binds to receptors oncell membranes of Gram Negative bacteria resulting in a change in itsstructure, making the cell wall more permeable.

Example 6: Dextran-Polymyxin Conjugate Synthesis

The polysaccharide-poloxamer nanoparticle as shown in Formula 3 may beformed by conjugating the PMB to the monomers prior to thepolymerization (as discussed in Examples 6 and 7) or by conjugating thePMB to the surface of the polysaccharide-poloxamer nanoparticle. Forexample, dextran acrylate (Example 2) may be dissolved in 20 ml water,cooled to 0° C., and 5-300 mg of 1-cyano-4-dimethylamino pyridiniumtetrafluoroborate (CDAP) will be added and mixed for 30 seconds. TEA(0.2M, 0.04 ml per 5 mg CDAP) will be added dropwise with vigorousstirring, and the entire reaction mixture is transferred to 80 ml of icecold ethanol containing 1 ml of 10N HCl. The dextran precipitates, andthe precipitate is removed by cold centrifugation at 1000 g, for 5 min,and solubilized in 20-50 ml of 0.25M Na-bicarbonate buffer at pH 9.0. Tothis mixture 600-1000 mg of PMB (either powdered or solubilized inwater) will be added and stirred for 24 hours at 8° C. The entirereaction mixture is then transferred to 50,000 molecular weight cut-offdialysis tubing and dialyzed against 0.05M pyrogen-free phosphate bufferfor 6-10 days.

Example 7: Alternate Dextran-Polymyxin Conjugate Synthesis

Dextran acrylate (Example 2) may be dissolved in water and reacted withsodium periodate in order to generate functional aldehyde groupsavailable for subsequent reaction. The thus activated dextran will thenbe dissolved in DMSO along with polymyxin-B and reacted to conjugateusing a commercially available hetero-bifunctional photoaffinitycross-linker such as p-azidobenzoyl-hydrazide (ABH, Pierce). Thissolution will be handled in the dark and allowed controlled periods ofexposure to UV light via Blak-Ray B100A. Polymyxin-B in double-distilledwater (0.5) is combined with the heterobifunctional covalent ioncrosslinking reagent azidohenzoyl hydrazide, 50 μL of 50 mM in DSMO.Following constant stirring in a 37° C. water bath for 60 min, thepreparation is exposed to light-flashes (5×3 sec) from a halogen lampsource. Preparation of Hydrogel involved limited oxidation under darkconditions with sodium periodate (Nal04 30 mM, pH 7.0, 1.0 mL). Duringthe final phase (II) of the semisynthesis procedure, thepolymyxin-azidohenzoyl hydrazide reactive intermediate complex wascombined with partially oxidized Hydrogel fractions and the resultingreaction mixture was stirred continuously at 24° C. for 30 minutes.

Example 8: Short Interfering Ribonucleic Acids (siRNAs)

siRNAs can be designed to target several debilitating diseases.Inhibiting a target protein using siRNA's can effectively down regulateeither the function of an individual gene or group of genes withouteliciting a toxic or an immune response. Post-transcriptional genesilencing occurs through RNA interference, where the double strandedRNAs (dsRNAs) are cleaved into 21-23 nucleotide fragments (i.e., shortinterfering RNA: siRNA). Cleavage occurs by a cellular endonuclease ofthe ribonuclease-Ill type called DICER. The short duplexed siRNA's areunwound by a helicase with the antisense strand becoming incorporatedinto the multi-component RNA-induced silencing complex (RISC). Thismoiety mediates sequence-specific gene silencing by cleaving the targetmRNA.

A siRNA phosphodiester backbone is anionically charged and naked siRNAdoes not pass through the cell membrane. The electrostatic repulsionbetween naked siRNA and the anionic cell membrane surface prevents nakedsiRNA endocytosis. Therefore, a delivery system is required forefficient transport and release. The most commonly used gene deliverysystems can be divided into biological (viral) and nonbiological(non-viral) systems. Each group has its own advantages and limitations.Biological carriers and viruses possess high efficiency in siRNAtransfer but are difficult to produce and may be toxic. Theselimitations favor non-biological systems for siRNA delivery.

Non-viral delivery systems including peptides, lipids (liposomes),dendrimers and polymers with cationic charges that interact with thenegatively charged siRNA through electrostatic interactions. A recentreview focused on use of precise polymer conjugates as nucleic aciddelivery and concluded that the materials for delivery of siRNA had tobe precise polymers, with defined site-specific conjugation strategiesthat provided multifunctional conjugates for nucleic acid transport.Dendrimers, defined peptide carriers, sequence-defined polyamidoaminesassembled by solid-phase supported synthesis, and precise lipopeptidesor lipopolymers have been characterized for pDNA and siRNA delivery.Conjugation techniques such as click chemistries and peptide ligationare available for conjugating polymers with functional transportelements such as targeting or shielding domains and for direct covalentmodification of therapeutic nucleic acids in a site-specific mode.However, the efficacy of RNAi in vivo depends upon efficient delivery ofthe intermediates of RNAi, such as short interfering RNA (siRNA).

Short inhibitory RNA's targeting the gram-negative LpxC gene.

Inhibiting target protein synthesis using siRNA's can effectively downregulate either the function of an individual gene or group of geneswithout eliciting a toxic or an immune response. Thus by inhibitingexpression of the LpxC gene using siRNA, bacterial cell wall synthesisis prevented and infectious sequalae will be prevented or aborted. Themechanism involves unwinding the short duplexed siRNA's by a helicasewith the antisense strand becoming incorporated into the multi-componentRNA-induced silencing complex (RISC). This moiety mediatessequence-specific gene silencing by cleaving the target mRNA. Severalsequences in the LpxC gene from both E. coli, P. aeruginosa and severalother gram-negative enteric bacteria have been identified. siRNA's willbe constructed from these sequences and employ the siRNA's to treatinfections induced by these and other strains of bacteria.

Exemplary siRNA's are described below.

siRNA #3 mRNA AGGGTGACGTCAAAGTGGATACG sense siRNA5′: GGUGACGUCAAAGUGGAUA 3′ Anti sense siRNA 3′: TdT CCACUGCAGUUUCACCUAU

Blast analysis indicates alignment complete alignment (100%) for thecoding sequence of the LpxC gene (UDP-3-O-acyl-N-acetylglucosaminedeacetylase) against multiple strains of Pseudomonas aeruginosa andshould prove effective against all P. aeruginosa.

Pseudomonas aeruginosa has become an important cause of infection,especially in patients with compromised host defense mechanisms. It isthe most common pathogen isolated from patients who have beenhospitalized longer than 1 week. It is a frequent cause of nosocomialinfections such as pneumonia, urinary tract infections (UTIs), andbacteremia. Pseudomonal infections are complicated and can be lifethreatening,

siRNA #4 mRNA: GACTTGAATCCACCGGTAGATTT Sense siRNA5′: CUUGAAUCCACCGGUAGAUdTdT Anti Sense siRNA 3′: TdTdGAACUUAGGUGGCCAUCUA

Blast analysis indicates efficacy against all strains of E. coli.Analyses shows further complete alignment against at least 21 strains ofSalmonella and several strains of Shigella, Klebsiella and Enterobacter.The most frequent bacterial cause of urinary tract infection (UTI) inadult women is Escherichia coli, which is part of the normal gut flora.This organism accounts for approximately 85% of community-acquired UTIsand 50% of hospital-acquired UTIs. Other common organisms includeKlebsiella pneumoniae, K. pneumoniae has been a recognized pulmonarypathogen since its discovery >100 years ago.

Klebsiella pneumoniae is among the most common gram negative bacteriaencountered by physicians worldwide. It is a common hospital-acquiredpathogen, causing urinary tract infections, nosocomial pneumonia, andintraabdominal infections. K. pneumoniae is also a potentialcommunity-acquired pathogen.

Enterobacter species, particularly Enterobacter cloacae and Enterobacteraerogenes, are important nosocomial pathogens responsible for variousinfections, including bacteremia, lower respiratory tract infections,skin and soft-tissue infections, urinary tract infections (UTIs),endocarditis, intra-abdominal infections, septic arthritis,osteomyelitis, and ophthalmic infections. Enterobacter species can alsocause various community-acquired infections, including UTIs, skin andsoft-tissue infections, and wound infections, among others.

Shigella bacteria produce toxins that can attack the lining of the largeintestine, causing swelling, ulcers on the intestinal wall, and bloodydiarrhea.

Salmonella enterica serovar Typhimurium, is the causative agent oftyphoid fever. Salmonella enterica serovar Typhimurium is the mostcommon cause of food poisoning by Salmonella species. Salmonellainfections are often fatal if they are not treated with antibiotics.

siRNA #5 mRNA: GAGCATGATGTACGGATTTCAAC Sense siRNA5′: GCAUGAUGUACGGAUUUCAdTdT Anti Sense siRNA 3′: TdTdCGUACUACAUGCCUAAAGU

Blast analysis indicates efficacy against all strains of E. coli.Further analyses show complete alignment against Salmonella and severalstrains of Shigella.

Polymyxins are cyclic polypeptide antibiotics. In addition to theirbacteriocidal properties they bind to the lipid A portion of endotoxinsand block their biologic properties. Low doses of polymyxin B have beenused therapeutically in burn patients to neutralize circulatingendotoxin consequent to burn injury. Low concentrations of polymyxinbound to a dextran-poloxamer nanogel will not present a toxic problem,in contrast to the therapeutic doses of Colistin, structurally relatedto polymyxin, currently in use. The lipopolysaccharides (LPS) of gramnegative bacteria function to provide membrane stabilization, integrityand confer resistance to host defenses. LPS is critical to theproliferation of gram-negative bacteria and disruption, mutation orremoval of LPS results in bacterial death. All gram negative bacteriaexhibit LPS in their outer membrane. LPS is composed of complexcarbohydrates and lipid A. The carbohydrates vary in structure andconfer the antigenic properties which distinguish different bacterialstrains. The Lipid A is highly conserved across strains and isresponsible for many of the pathogenic, immunologic and pyrogeniceffects of gram negative bacteria The ubiquitous nature of LPS makes itan attractive target for treatment of nearly all Gram-negative bacterialstrains. Lipid A is a critical structural component of LPS. It functionsto anchor LPS to the cell membrane and it is structurally bound to thecore polysaccharide.

The synthesis of the KDO lipid A complex is under control of a series ofseveral constitutively expressed enzymes. These are LpxA, LpxC, LpxD, LpH, LpxB, LpxK, LpxL and LpxM. Not all bacteria possess all of theseenzymes but the first four are commonly expressed in gram negativebacteria. Lpx C is a zinc-dependent deacylase(UDP-(3-0-(R-3-hydroxymyristoyl))-N-acetylglucosamine deacylase). It isthe first committed step in Lipid A biosynthesis and has been shown tobe essential for growth of E. coli. Indeed several labs have focused onthe antibacterial properties of pharmacologic agents which inhibit LpxCenzymatic activity, (antimicrob. Agents Chemother. 50: 2178, 2006, Curr.Pharm Biotechnol. 9; 9, 2008) Antimicrobial resistance mechanisms havebeen reported for all known antibacterial drugs that are currentlyavailable. Thus the inventors have focused their attention of strategieson the development of new and more effective agents that bypass thethreat of antibiotic resistance by inhibiting the expression of the LpxCgene.

Polymyxin B conjugated to an embodiment of the poloxamer-polysaccharidehydrogel produces a safe, effective, in vivo nanotherapeutic platformfor targeting and killing gram negative bacteria through controlleddelivery of nondrug antibacterial compounds, e.g., siRNA, PVP-I, Silver.

TABLE 1 Site Prodrug Mode of Action Cell Polymyxins Phospholipidsmembrane

Additional Gram Negative Targeting Prodrug Conjugations for DextranComonomer of Polysaccharide-Poloxamer to Deliver Antibiotics

Penicillin, Cephalosporin, and Glycopeptide are r-lactam antibioticsthat inhibit bacterial cell wall biosynthesis and afford an opportunityas potential conjugates of the polysaccharide-poloxamer drug deliverysystem to target the multiple drug resistant bacteria. The otherantibiotics act in the cell and to be effective these antibiotics mustpenetrate the cell wall. Aminoglycosides, for example, have to beactively transported across the bacterial cell membrane. Glycoproteins,for example, vancomycin and teicoplanin, are unable to penetrate theouter membrane of gram-negative organisms and thus have restrictedactivity against these organisms. Conjugation to apolysaccharide-poloxamer nanogel may allow these antibiotics to passthrough the cell wall, be released, and effectively kill the bacteria.

TABLE 2 Site ProDrug Mode of Action Cell wall Penicillins TranspeptidaseCephalosporins Transpeptidase Glycopeptides Acyl-D-alanyl-D-alanineCarbapenem

Example 9: Amine Conjugation on Polysaccharide-Poloxamer Hydrogel forCationic Charged Nanogel Delivery

Amines may also be conjugated to the polysaccharide-poloxamernanoparticles or hydrogel. Cationically charged compounds may then bechemically attached to the nanoparticles or hydrogel. Nanoparticles witha primary amine at the surface promote higher rates of phagocyticuptake. To produce a charged Polysaccharide-poloxamer nanoparticle, anamine can be conjugated to dextran prior to polymerization (as discussedin Example 9) or the surface of the nanoparticle may be functionalizedafter polymerization.

To incorporate amine group into dextran-acrylate (Example 2),dextran-acrylate may be further reacted with 3-CHLOROPROPYLAMINEHYDROCHLORIDE in the presence of triethylamine. An example ofdextran-acrylate-propylamine synthesis is given here:

Pre-dried dextran-acrylate (2.0 g) will be dissolved in anhydrous DMSOunder nitrogen gas at room temperature. Triethylamine (11.2 ml) was theninjected into the above solution. Meanwhile, 3-chloropropylaminehydrochloride (4.8 g) will be dissolved in DMSO and then added to theabove solution drop wise, and stirred for 5 hours at 50° C.Dextran-acrylate-propylamine will be obtained by precipitating thefiltered solution into excess cold isopropyl alcohol. The product wasfurther purified three times by dissolution/precipitation with DMSO/coldisopropyl alcohol. The final product was dried overnight at roomtemperature under vacuum before further use.

“Charged”-siRNAs or other charged medicaments or targeting agents maythen be chemically attached to or encapsulated in the aminefunctionalized polysaccharide-poloxamer nanoparticle produced from theamine functionalized dextran-acrylate and delivered as targeted cargo tobacteria.

Example 9: Antibody Conjugated Polysaccharide-Poloxamer Nanoparticlesfor Treatment for Treatment of Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is an aggressive breast cancerphenotype characterized by lack of expression of estrogen receptor (ER)and progesterone receptor (PR), as well as the absence of overexpressedhuman epidermal growth factor receptor-2 (HER-2) (1. de Ruijter et al).As noted below, this threatening disease is far reaching in its effects.About 15% of breast cancer patients are diagnosed with triple negativebreast cancer. An estimated 1 million cases of breast cancer arediagnosed annually worldwide. Of these, approximately 170,000 are of thetriple-negative (ER-/PR-/HER2-) phenotype. Of these TNBC cases, about75% are “basal-like.”

TNBC is generally accepted as a clinical surrogate for basal-like breastcancer. All basal-like breast cancers are not triple negative however.This phenotype is associated with an early age of cancer onset, highchance of presentation with metastases and high proliferative index(Nofech-Mozes et al). The prognosis of patients with this type of tumoris very poor because of non-responsiveness to hormonal therapy or poorresponse to the therapy of choice in breast cancer—Tamoxifen. Hence,there is an urgent and unmet need for efficacious therapeutics to treatTNBC. Anti-EGFR therapy has been increasingly recognized as an importanttreatment for TNBC patients and is being evaluated in advanced clinicaltrials for patients with metastatic TNBC. High expression of epidermalgrowth factor receptor (EGFR) induces erroneous development andunrestricted proliferation in a number of human malignancies, includingbreast cancer and also prostate cancer. This receptor has long beenconsidered as a potential target for the treatment of a number of cancertypes. EGFR mRNA is detected more frequently and at higher levels inbasal-like breast cancers. Antibody dependent cellular cytotoxicity isrecognized as prominent cytotoxic mechanism for therapeutic monoclonalantibody. There are a number of monoclonal antibodies (mAbs) currentlyavailable on the market for cancer treatment and a plethora beingevaluated in clinical trials exhibiting mixed therapeutic outcomes. Foran optimum therapeutic response, monoclonal antibodies (mAbs) shouldexhibit a sufficiently long half-life to interact with the target tissueeffectively, have the capability to get internalized in the tumorinterior, have no inducement of an immune response, and deliversufficient potency (16. Manuel et al). Unfortunately, most of themarketed mAbs do not fulfill all of these requirements, thus providing asuboptimal therapeutic response.

Nanotechnology is an area of manipulation/construction of structures ina nanometer size range. The chemical/physical properties of theconstruct can radically change at this level, which can be exploited todeliver the antibody to uncharted destinations efficiently and alsocarry more antibodies precisely to the site of action which will elicitgreater effect. An antibody capable of identifying tumor antigens can beanchored on the surface of the nanocarriers to increase the targetingefficiency, thereby increasing drug accumulation in the tumor tissue.These antibody conjugated nanocarriers can provide long circulation andsignificantly higher tumor accumulation properties (due to enhancedpermeability retention effect) which yield significant improvements intherapeutic efficacy.

Some of the other examples of marketed antibody conjugates are withcytotoxic drugs (Mylortag®) or radioisotopes (ProstaScint®). However, todate, there are no commercial antibodies conjugated to nanoparticlesavailable in the cancer treatment regimen. The available data suggests asignificant edge of antibody conjugated nanoparticles in cancer therapyin terms of efficacy and a reduction in toxicity. The treatment of TNBCpresents a momentous challenge to the oncologist often faced withlimited therapeutic options coupled with aggressive and unresponsivetumors. Thus, development of an efficient therapeutic system foreffective treatment modality of TNBC is an urgent need. This could beaddressed by conjugating clinically relevant antibody to nanoparticles.

A therapeutic modality for the treatment of TNBC in the form of EGFRantibody conjugated to polysaccharide-poly(epoxide) nanoparticles willbe developed to yield significant therapeutic benefit over currenttherapies.

A revolutionary new biocompatible hydrogel drug delivery platform basedon a polymeric network of cross-linked polysaccharide monomers andepoxide monomers will be used to form nanoparticles. In a particularembodiment, a hydrogel synthesized from crosslinking a modified dextran(a natural polymer) and another FDA approved polymer (Pluronic® F-127)also modified to facilitate (esterification) UV crosslinking will createthe new copolymer hydrogel. Pluronic® F-127 is biodegradable and alsoprovides thermally responsive properties when incorporated into thenanoparticle. The elevated and narrow range of human body temperatureoffers an ideal trigger for thermal responsive hydrogels that employ theblock copolymer Pluronic® F-127 obtained from BASF and offers “tunableoptimization” per application for “tailored” controlled therapy. Thishydrogel is amenable for conversion to nanoparticles either by lop downor bottom up approaches with narrow particle size distribution.

An alternative to the UV crosslinked hydrogel procedure is production ofthe hydrogels by inverse miniemulsion polymerization and crosslinkedin-situ by free radical mechanisms. The advantage of this approach is alesser energy demand of the process and the elimination of the need forhigh energy equipment. The polysaccharide-poloxamer platform (inspecific embodiments, dextran-pluronic F-127 nanoparticle) also offers“tunable” optimization of the nanogels for different applications for“tailored” controlled therapy. Embodiments of the nanoparticle and/orhydrogel is amenable for formation to nanoparticles either by a “topdown” or a “bottom up” approach to produce nanoparticles of narrowparticle size distribution. A bottom-up approach constructsnanoparticles from basic building blocks like atoms or molecules as in aminiemulsion polymerization process. A top-down approach producesnanoparticles from larger materials from physical processes such as, forexample, grinding or milling or through chemical-based processes (bondbreaking).

EGFR antibody conjugated nanoparticles derived from polysaccharides andpoloxamers would yield better therapeutic response in TNBC due to thefollowing:

-   -   These mAb conjugated nanoparticles would exhibit higher        accumulation at the tumor site due to an enhanced permeability        effect;    -   The hydrophilic surface of the nanoparticles would render them        long circulating (Karmali et al); and    -   Biocompatible cargo is not expected to yield any toxicity or        immune reactions (van Manen et al).

These properties of embodiments of the polysaccharide-poly(epoxide)nanoparticles may result in better treatment of TNBC. Commerciallyavailable or clinically evaluated EGFR mAb would be preferred, as itwould propel the development cycle significantly and shorten the timefor market entry. The EGFR mAb may be selected from, but not limited to,Cetuximab (Harding et al, Vincenzi et al), Panitumumab (Ferraro et al,Carteni et al) or Zalutumumab (Rivera et al). The nanotechnology willyield viable options to otherwise non responsive and aggressive TNBC.Hence, embodiments of the polysaccharide-poly(epoxide) will yield atherapeutically efficient, commercially viable niche formulation totreat TNBC with a scope of extending the indications to other cancertypes exhibiting EGFR overexpression such as prostate cancer.

Example 9A Formulation of Polysaccharide-Poloxamer Nanoparticles

In this embodiment, a “bottom up” technique for formation ofnanoparticles using inverse emulsion polymerization will be used toproduce the polysaccharide-poloxamer nanoparticles. This technique hasbeen developed and optimized. The inverse emulsion polymerizationtechnique selected for the production of nanoparticles is simple,versatile and easy to scale-up. The size of the macromonomer inverseemulsion droplets can be manipulated by varying emulsifierconcentration. In this embodiment, polysaccharide-poloxamer nanogels ofdesired size may be produced with the controlled droplets of watersoluble dextran-acrylates UV crosslinked to Pluronic® F-127 diacrylatesin solution. Photopolymerization proceeds very fast but irradiation maybe allowed to proceed for an extended time, such as for 1 hour to ensuresubstantially complete polymerization.

Other polysaccharides can be cross-linked to Pluronic® F-127 or otherpoloxamers (or more generally poly(epoxides), in inverse emulsionphotopolymerization, provided they are soluble in water and that themonomers contain, or are functionalized with, polymerizable groups.

Dextran-pluronic F-127 nanoparticles, other polysaccharide-poloxamer, orpolysaccharide-poly(epoxide) based hydrogels can be obtained via inverseemulsion photopolymerization. Nanoparticle size can be controlledthrough choice of emulisifer(s), monomer and emulsifier concentration,and polymerization process conditions.

Example 9B Conjugation of EGFR Antibody to Dextran-Pluronic F-127Nanoparticles, Other Polysaccharide-Poloxamer, orPolysaccharide-Poly(Epoxide) Nanoparticles

In this embodiment, EGFR antibody will be conjugated to the surface ofdextran-pluronic F-127 nanoparticles after formation of thenanoparticles. Alternatively, the EGFR antibody may be conjugated to themonomers with subsequent formation of nanoparticles. A mAb within thenanoparticle matrix would elicit therapeutic response after degradationof the nanoparticle. The surface pendent mAb recognizes the targetreceptor and attaches to them to elicit bioactivity of the cell.

The Fc-directed conjugation of the antibody molecules would be madethrough reductive amination coupling between the free amino groups inthe Fc-region of the antibody and reactive aldehyde groups. To createreactive aldehyde groups on the nanoparticles surface, oxidation ofdextran may be carried out under mild conditions using sodium iodate anda fixed concentration of dextran-pluronic F-127 nanoparticles. Thisreaction may be performed in the dark and under an inert atmosphere. Theoxidation reaction will be quenched by the addition of ethylene glycol.The nanoparticles will be purified by dialysis. To this, differentconcentrations of EGFR antibody will be added and incubated. Thisconjugated structure will be stabilized by reduction using sodiumborohydride. Finally, the nanoparticles will be purified andconcentrated using spin filter (Rezaeipoor et al). Optionally,fluorescent tagged mAB will also be used as marker for cellular uptakeand trafficking study.

Example 9C Determination of Antibody Concentration on the Nanoparticle

The final antibody/nanoparticle ratio will be determined using abicinchoninic acid (BCA) assay (Protein Quantitation Assay, Pierce).

Example 9D Physiochemical Characterization of mAB ConjugatedDextran-Pluronic F-127 Nanoparticles

The size and zeta potential of dextran-pluronic F-127 nanoparticles willbe evaluated by dynamic light scattering technique. This technique willalso be used to determine any changes in the nanoparticles'characteristics due to mAb conjugation. The shape of nanoparticles willbe accessed by transmission electron microscopy after negative stainingwith uranyl acetate or phosphotungstic acid or osmium tetroxide. Surfacecharacteristics of mAb conjugated dextran-pluronic F-127 nanoparticleswill be done to evaluate the effect of the conjugation process.

Example 9E In Vitro Cell Culture Studies

A number of different cell lines will be used to evaluate the developedmAb conjugated dextran-pluronic F-127 nanoparticles. The cell linesevaluated will include human breast cancer cell lines MDA-MB-468 (TNBC,EGFR-positive), SKBR-3 (EGFR-positive), BT-474 (EGFR-positive), andMCF-7 (EGFR-negative). The dextran-pluronic F-127 nanoparticlesconjugated with different mAb concentrations, naked mAb, andunconjugated dextran-pluronic F-127 nanoparticles will be evaluated indifferent cell culture studies such as proliferation assay, cell cycleassay, and western blot analysis and the cell uptake will be evaluatedby (fluorescent conjugated mAb) flowcytometry, confocal microscopy, etc.

These studies will be designed to check retention of bioactivity of mAbafter the conjugation process, and optimization of mAb concentration onthe dextran-pluronic F-127 nanoparticles to yield optimal bioactivity.In vitro and in vivo evaluation of three prototype EGFR monoclonalantibody conjugated dextran-pluronic F-127 nanoparticles will beperformed. The primary objective of these studies is to determineanti-tumor activity of these novel reagents with a model of breastcancer that is EGFR positive and is ER- PR- and HER2-. In this model wewill determine whether the antibody targeting EGFR conjugated todextran-pluronic F-127 superior efficacy compared to the unconjugatedantibody because it would have better bioavailability and deliver betterproperties than conventional formulations, which would in turn translateto better tumor growth inhibition.

Prior to in vivo efficacy testing of these novel nanoparticles it willbe important to determine their safety upon intravenous delivery toimmunocompromised athymic nude mice. Also, prior to evaluating in vivotolerability of the antibody conjugated nanoparticles, we will testtheir toxicity in vitro. First, we will perform the MTT assay with theMDA-MB-468 breast cancer cells that will be utilized in the in vivoefficacy study. Hemolysis and micronucleus test of genotoxicity will beperformed.

Example 9F In Vitro Toxicology Studies

Evaluation of toxicity of dextran-pluronic F-127 nanoparticles is animportant step in development of any nanotechnology based therapeuticagent. (Arora et al, Kroll et al, 2009, Kroll et al, 2012). In vitromodel systems provide a rapid and effective means to assessnanoparticles for specific toxicological endpoints. These studies allowfor elucidation of the mechanism of interaction of nanoparticles withcells. Hence in vitro studies can be effectively used to establishspecific toxicological profiles of developed nanoparticles and wouldhelp to design the protocol of in vivo studies.

Using the established protocol (Zhang et al), the MTT assay willdetermine the effect of two-three prototypes antibody conjugatednanoparticles on MDA-MB-468 cell viability and metabolic activitymeasured by the reduction of the tetrazolium salt MTT to insolubleMTT-formazan. The unconjugated dextran-poloxamer nanoparticles will alsobe tested as control. Moreover the hemolysis (Zhang et al, Yu et al) andmicronucleus genotoxic test (Gonzalez et al) will measure differentcytotoxicity endpoints of the antibody conjugated dextran-poloxamernanoparticles.

The results from these studies will allow us to select the optimalconcentration of the antibody formulation that will be tested in the invivo efficacy and compared with the naked antibody, control unconjugatednanoparticles and saline.

Example 9G In Vivo Tolerability Study in Female Athymic Nude Mice

In this study mice will receive treatment intravenously twice a week,and will be carefully observed every day for at least two weeks for anysign of distress, abnormal behavior, body weight loss, morbidity, andmortality. Gross examination of organs will be done. The results fromthis study will be useful to assess safety of antibody conjugatednanoparticles in mice prior to testing their efficacy.

Example 9H In Vivo Efficacy Study in Female Athymic Nude Mice

The MDA-MB-468 tumor cells implanted in mice for this study will befirst transfected with the luciferase lentiviral particles and implantedinto the mammary fat pad of five mice to ensure the tumors grown in vivoretain bioluminescence. Then the luciferase-labeled tumor cells will beharvested from in vitro cultures and implanted with 50% matrigel in themammary fat pads of nude mice (left side, 5×106 per mouse). When thetumors reached an average size of at least 130 mm3 the mice will berandomized and distributed into 5 groups of 10 mice/each group withsimilar tumor size and bioluminescent signal measured by the LuminaInstrumentation after intraperitoneal injection of D-luciferin (15mg/ml, 200 μl). The five treatment groups are two prototype mABconjugated polysaccharide-poly(epoxide), polysaccharide-poloxamer, ordextran-pluronic F-127 nanoparticles formulations, the unconjugatedmonoclonal antibody, the unconjugated nanoparticles, and saline.Treatment will be delivered intravenously twice a week for 4 weeks. Thetumors will be calipered one a week or more often, and imaged with theLumina Instrument once a week; at the end of the study, the mice will beeuthanized three days after last treatment and the tumors will beharvested and fixed in 10% formalin for histology and analysis of tissuemorphology. (Inoue et al, 33. Mitsunaga et al).

Example 9I Animal Models

The tumor targeting efficacy will be evaluated by non-invasive imagingtechniques. The toxicology profile of the formulation will be generatedin a suitable animal model. The process for preparing the mAb conjugatedpolysaccharide-poloxamer nanoparticles will be optimized in view ofscale up activities. Studies to convert the optimized nanoparticulateformulation into patient administrable dosage form will be initiated.Also, container closure selection study will be initiated. Analyticalmethods used in formulation evaluation will be validated.

Example 10: Mannosylation of Dextran-Pluronic F-127 Hydrogel

Tuberculosis (TB) is the leading cause of death in the world from abacterial infectious disease. The disease affects 1.8 billion peopleyearly, equal to one-third of the entire world population.

The treatment of tuberculosis requires long-term antibiotic therapy.Because administration of a single drug often leads to the developmentof a bacterial population resistant to that drug, effective regimens forthe treatment of TB must contain multiple drugs to which the organismsare susceptible. Active tuberculosis, particularly if it's adrug-resistant strain, will require several drugs at once. The mostcommon medications used to treat tuberculosis include, but are notlimited to, Isoniazid, Rifampin (Rifadin, Rimactane), Ethambutol(Myambutol), and Pyrazinamide.

Mycobacterium tuberculosis (MTB) is the etiologic agent of tuberculosisin humans. Humans are the only reservoir for the bacterium. Targetedantibiotic therapy improves the efficacy of treatment by concentratingthe drugs close to mycobacterium. Once attached to a macrophage, thecausative agent of tuberculosis, MTB avoids cellular defenses and usesthe cell to replicate. The high concentration of lipids in the cell wallof Mycobacterium tuberculosis has been associated with impermeability tostains and dyes, resistance to many antibiotics, resistance to lethaloxidations and survival inside of macrophages. Polysaccharide-poloxamertargeting of MTB is accomplished by using mannose as a homing device.

Mannosylation of the dextran monomer enables the multidrug releasingdelivery system, Polysaccharide-poloxamer, to target the macrophage.Ref. Clemens, D. L, and M. A. Horwitz. 1996. The Mycobacteriumtuberculosis phagosome interacts with early endosomes and is accessibleto exogenously administered transferrin. J. Exp. Med. 184.1349-1355.

The development of a polysaccharide-poloxamer delivery vehicle is basedon dextran linked to mannose, to target the macrophage, for multidrugtherapeutic release into the phagosomal vacuole. Water-solublepolysaccharide-poloxamer hydrogel can accommodate mannose homingresidues (endocytosis by the target cell via a specific receptor) thusincreasing the affinity for the target. Ref. Kėry, V., J. J. F.Krepinsky, C. D. Warren, P. Capek, and P. D. Stahl. 1992. Targeting isaccomplished through ligand recognition by purified human mannosereceptors. Arch. Biochem. Biophys. 298:49-55.

To synthesize MTB targeted nanogels and/or nanoparticles, the monomerssuch as, but not limited to, the dextran monomer of thepolysaccharide-poloxamer drug delivery carrier may conjugated to mannosewhich acts as a targeting agent to direct multiple antibacterialreleasing Polysaccharide-poloxamer into macrophages.

Example 10A Dextran NO₂—NH₂ Conjugation

Dextran (Pharmacia, Uppsala, Sweden) was can be purified to getfractions with weight-average molar masses of 64,000, 90,000, or 95,000g/mol and with a weight-average molar mass/number-average molar massratio of 1.2.

Poloxamer: Pluronic F-68 BASF.

For this purpose, norfloxacin has been linked to mannosylated dextranusing a peptide spacer arm. This conjugate shows more efficacy againstmycobacterium than plain norfloxacin.

Example 11: Methods of Producing a Polysaccharide-Poloxamer Nanoparticle

The polysaccharide and poloxamer may be cross-linked with anypolymerization process or appropriate cross-linking reaction includingradical polymerizations, emulsion polymerizations, inverse miniemulsionpolymerization, controlled polymerization, UV initiated cross-linking,e-beam curing, ionic gelation polymerization or other polymerizationprocess. The process, component concentrations and the processparameters may have a significant effect on the properties of thehydrogel or nanoparticles such as, but not limited to, rates ofdiffusion of the pharmaceuticals out of the hydrogel membrane.

The ratio of polysaccharide to poly(epoxide), the molecular weight ofthe polysaccharide and/or the poly(epoxide), the chemical composition ofthe polysaccharide and/or the poly(epoxide), the relative lengths of theABA block of the poloxamer, the degree of self-organization prior tocross-linking, the cross-linking functionality, as well as other factorsmay affect the physical and chemical properties of the hydrogel. Forexample, the mechanical strength of the hydrogel or polymeric networkcan be adjusted by more or less polysaccharide (in some embodiments,dextran) which will produce a different hydrogel or polymeric networkwith different mechanical strength and a different time controlleddelivery of a drug for drug delivery applications. The chemicalcomposition and size are important factors, which are indicative of thecapability of the particles to penetrate into biological cells.

An inverse miniemulsion polymerization process for the production ofnanoparticles is preferred because it is simple, versatile and easy toscale-up. A number of different monomers may be included in thecross-linked, hydrogel network of the nanoparticles, providing aflexible way of regulating material properties and introducingfunctionality, incorporating electrostatically charged and reactivefunctional groups by copolymerization of appropriate monomers. Inverseemulsion photopolymerization is a controllable method for preparing“more defined” nanoparticles. The aqueous macromonomer nano-droplets are“stabilized” by a cross-linking polymerization of acrylic derivatives,which preserves the structure of the nanoparticles. The nanoparticlesproduced present a capacity (nanodomain) of incorporating hydrophobicdrugs.

In one embodiment, the aqueous phase, containing eosin Y (sensitizer),triethanolamine (initiator) and Pluronic F-127 diacrylate mixed withdextran diacrylate, is dispersed in hexane by sonication with theutilization of the powerful surfactant, Span 65, in the oil-to-waterprotocol. After photopolymerization, nanoparticles can be by removedfrom the hydrophobic emulsifier through repeated n-hexane washing.

The surfactant, Span65, is dissolved in hexane by sonication. Nonionicsurfactants such as Span and/or Tween may be used in the reaction media.An aqueous solution of dextran acrylate, Pluronic F-127 diacrylate,triethanolamine, and eosin Y is added to the oil phase (oil-to-waterweight ratio=65/35) and an inverse emulsion can be formed. The inverseemulsion can be illuminated with an Ar ion laser for 1 hour, at roomtemperature, under magnetic stirring (400 rpm). After illumination, theinverse emulsion can be poured into centrifuge tubes containing n-hexaneand water. The aqueous phase is extracted with n-hexane to remove thesurfactant and then dialyzed against water to remove the initiator andnon-reacted macromonomers.

Again, conjugation of targeting agent(s) onto the alcohol reactivegroups of the modified dextran (polysaccharide) comonomer may be a firststep in the production process of targeting therapeutic nanoparticles.

Inverse emulsion polymerization process has the ability to “controlnanoparticle size” distribution. The size of the macromonomer inverseemulsion droplets decreased with increasing emulsifier concentration.Polysaccharide-poloxamer nanogels of desired size are produced with thecontrolled droplets of water soluble dextran acrylates UV cross-linkedto Pluronic® F-127 diacrylates in solution. Photopolymerization proceedsvery fast but irradiation may be carried out for an extended period toensure the desired degree of polymerization. Other polysaccharides canbe cross-linked to Pluronic® F-127 in inverse emulsionphotopolymerization, provided they are soluble in water and that theycontain, or are functionalized with, polymerizable groups. The “stable”colloidal state of the resulting nanoparticles is maintained even afterfreeze drying.

Further, the nanogels and nanoparticles may be effective in cancertreatment. Progress in fundamental cancer biology has not yet been metby a comparable advancement in its clinical treatment. A fundamentalreason for this discrepancy is the inability to selectively reach andeliminate tumor tissue with marginal damage to healthy organs, cancercell targeting by polysaccharide-poloxamer drug delivery systems aims atincreasing selectivity and overcoming biological barriers, whiletransporting higher drug amounts.

Active targeting is accomplished by attachment of specific molecules onthe carrier's surface, which enhance the binding and interactions withantigens or receptors expressed on specific cell populations. Targetingligands explored for cancer therapy include antibodies and antibodyfragments which can be conjugated to Polysaccharide-poloxame forspecific active targeted drug delivery.

A major class of chemotherapeutics currently used in clinical practiceare the anthracycline molecules. Doxorubicin is probably the most knownmember of the anthracycline family. These potent anti-proliferativeagents are a typical example of drugs whose efficacy is constrained bynon-specific toxicities and would therefore benefit by thepolysaccharide-poloxamer targeted drug delivery approach

Immune response and biodegradability issues are a significant concernfor drug delivery devices, as well as issues relating to drug targetingand controlled drug release. Consequently, there is an immediate needfor a comprehensive answer to these problems. A dextran-poloxamercopolymer nanoparticle (nanogel) offers a universal platform thatguarantees a safe, sustained, and controlled drug delivery system.

The thermal response feature of the poloxamer (Pluronic® F-127)comonomer component and the controlled release capability (various ratiocompositions of dextran-poloxamer) of polysaccharide-poloxamer nanogelsenable development of more effective therapeutic nanoparticles. Withcontinuous advances in identifying new biomarkers and associatedtargeting ligands it will be increasingly feasible to develop targetedand controlled release nanoparticle products as promising candidates forclinical translation.

Nanoparticles comprised of polysaccharides and poloxamers offerexcellent nanocarrier capabilities. Particularly, the Dextran-Pluronicnanogels exhibit ideal characteristics and features desired in a drugdelivery system. They are nontoxic, nonimmunogenic, nonantigenic, andbiodegradable. For targeting, especially, they present a great number ofhydroxyl groups for conjugation of prodrugs, enzymes, heavy metals(e.g., Fe) and small molecules, for example.

REFERENCES

-   Adler M J, Dimitrov D S. Therapeutic antibodies against cancer.    Hematol Oncol Clin North Am. 2012 Jun. 26(3):447-81.-   Arora S, Rajwade J M, Paknikar K M. Nanotoxicology and in vitro    studies: the need of the hour. Toxicol Appl Pharmacol. 2012 Jan. 15;    258(2):151-65.-   Carteni G, Fiorentino R, Vecchione L, Chiurazzi B, Battista C.    Panitumumab a novel drug in cancer treatment. Ann Oncol. 2007 Jun.    18 Suppl 6:vi16-21.-   Corkery 8, Crown J, Clynes M, O'Donovan N. Epidermal growth factor    receptor as a potential therapeutic target in triple-negative breast    cancer. Ann Oncol. 2009 May; 20(5):862-7.-   de Ruijter T C, Veeck J, de Hoon J P, van Engeland M, Tjan-Heijnen    V C. Characteristics of triple-negative breast cancer. J Cancer Res    Clin Oncol. 2011 February; 137(2):183-92.-   Del Mastro L, Lambertini M, Bighin C, Levaggi A, D'Alonzo A, Giraudi    S, Pronzato P. Trastuzumab as first-line therapy in HER2-positive    metastatic breast cancer patients. Expert Rev Anticancer Ther. 2012    Nov. 12(11):1391-405.-   Duffy M J. Tumor markers in clinical practice: a review focusing on    common solid cancers. Med Princ Pract. 2013; 22(1):4-11.-   Ferraro D A, Gaborit N, Maron R, Cohen-Dvashi H, Porat Z, Pareja F,    Lavi S, Lindzen M, Ben-Chetrit N, Sela M, Yarden Y. Inhibition of    triple-negative breast cancer models by combinations of antibodies    to EGFR. Proc Nat Acad Sci USA. 2013 Jan. 29; 110(5):1815-20.-   Gonzalez L, Sanderson B J, Kirsch-Volders M. Adaptations of the in    vitro MN assay for the genotoxicity assessment of nanomaterials.    Mutagenesis. 2011 Jan. 26(1):185-91.-   Greenberg S, Rugo H S. Challenging clinical scenarios: treatment of    patients with triple-negative or basal-like metastatic breast    cancer. Clin Breast Cancer. 2010 Sep. 10 Suppl 2:520-9-   Harding J, Burtness B. Cetuximab: an epidermal growth factor    receptor chemeric human-murine monoclonal antibody. Drugs Today    (Barc). 2005 February; 41(2):107-27-   Inoue S, Patil R, Portilla-Arias J, Ding H, Konda B, Espinoza A,    Mongayt D, Markman J L, Elramsisy A, Phillips H W, Black K L, Holler    E, Ljubimova J Y. Nanobiopolymer for direct targeting and inhibition    of EGFR expression in triple negative breast cancer. PLoS One. 2012;    7(2):e31070-   Karmali P P, Chao Y, Park J H, Sailor M J, Ruoslahti E, Esener S C,    Simberg D. Different effect of hydrogelation on antifouling and    circulation properties of dextran-iron oxide nanoparticles. Mol    Pharm. 2012 Mar. 5; 9(3):539-45.-   Kim E M, Jeong H J, Jeong M H, Lee C M, Cheong S J, Kim D W, Lim S    T, Sohn M H, dextran-conjugated vascular endothelial growth factor    receptor antibody for in vivo melanoma xenografted mouse imaging.    Cancer Biother Radiopharm. 2012 Mar. 27(2):141.8.-   Kroll A, Pillukat M H, Hahn D, Schnekenburger J. Current in vitro    methods in nanoparticle risk assessment: limitations and challenges.    Eur J Pharm Biopharm. 2009 June; 72(2):370-7.-   Kroll A, Pillukat M H, Hahn D, Schnekenburger J. Interference of    engineered nanoparticles with in vitro toxicity assays. Arch    Toxicol. 2012 July; 86(7):1123-36.-   Luedke E, Jaime-Ramirez A C, Bhave N, Carson W E 3rd. Monoclonal    antibody therapy of pancreatic cancer with cetuximab: potential for    immune modulation. J Immunother. 2012 June; 35(5):367-73.-   Manuel Arruebo, Mónica Valladares, and África González-Fernández,    “Antibody-Conjugated Nanoparticles for Biomedical Applications,”    Journal of Nanomaterials, vol. 2009, Article ID 439389, 24 pages,    2009, doi:10.1155/2009/439389-   Mendelsohn, The epidermal growth factor receptor as a target for    cancer therapy. Endocrine-Related Cancer 2001:8 3-9-   Mitsunaga M, Nakajima T, Sano K, Kramer-Marek G, Choyke P L,    Kobayashi H. Immediate in vivo target-specific cancer cell death    after near infrared photoimmunotherapy. BMC Cancer. 2012 Aug. 8;    12:345-   Nielsen T O, Hsu F D, Jensen K et al. Immunohistochemical and    clinical characterization of the basal-like subtype of invasive    breast carcinoma. Clin Cancer Res 2004; 10: 5367-5374.-   Nofech-Mozes S, Trudeau M, Kahn H K, Dent R, Rawlinson E, Sun P,    Narod S A, Hanna W M. Patterns of recurrence in the basal and    non-basal subtypes of triple-negative breast cancers. Breast Cancer    Res Treat. 2009 November; 118(1):131-7.-   Overdijk M B, Verploegen S, van den Brakel J H, Lammerts van Bueren    J J, Vink T, van de Winkel J G, Parren P W, Bleeker W K. Epidermal    growth factor receptor (EGFR) antibody-induced antibody-dependent    cellular cytotoxicity plays a prominent role in inhibiting    tumorigenesis, even of tumor cells insensitive to EGFR signaling    inhibition. J Immunol. 2011 Sep. 15; 187(6):3383-90.-   Patil R R, Guhagarkar S A, Devarajan P V. Engineered nanocarriers of    doxorubicin: a current update. Crit Rev Ther Drug Carrier Syst.    2008; 25(1):1-61.-   Peraldo-Neia C, Migliardi G, Mello-Grand M, Montemurro F, Segir R,    Pignochino Y, Cavalloni G, Torchio B, Mosso L, Chiorino G.    Aglietta M. Epidermal Growth Factor Receptor (EGFR) mutation    analysis, gene expression profiling and EGFR protein expression in    primary prostate cancer. BMC Cancer. 2011 Jan. 25; 11:31.-   Perou C M, Sorlie T, Eisen M B et al. Molecular portraits of human    breast tumours. Nature 2000; 406: 747-752.-   Pillay V, Gan H K, Scott A M. Antibodies in oncology. N Biotechnol.    2011 Sep. 28(5):518-29.-   Rezaeipoor R, John R, Adie S G, Chaney E J, Marjanovic M, Oldenburg    A L, Rinne S A, Boppart S A. Fc-directed antibody conjugation of    magnetic nanoparticles for enhanced molecular targeting. J Innov Opt    Health Sci. 2009 Oct. 1; 2(4):387-396.-   Rivera F, Salcedo M, Vega N, Blanco Y, López C. Current situation of    zalutumumab. Expert Opin Biol Ther. 2009 May; 9(5):667-74.-   Tischkowitz M, Brunet J S, Begin L R et al. Use of    immunohistochemical markers can refine prognosis in triple negative    breast cancer. BMC Cancer 2007; 7: 134.-   van Manen H-J, van Apeldoorn A A, Verrijk R, Blitterswijk CAv, Otto    C, Intracellular degradation of microspheres based on cross-linked    dextran hydrogels or amphiphilic block copolymers: A comparative    Raman microscopy study. Int J Nanomedicine. 2007 Jun. 2(2): 241-252.-   Vincenzi B, Zoccoli A, Pantano F, Venditti O, Galluzzo S. Cetuximab:    from bench to bedside. Curr Cancer Drug Targets. 2010 Feb.    10(1):80-95.-   Yeoman, Roy R, Fox, Adrian S, Sun, Guoming, Polymers And Hydrogels    WO/2013/058778-   Yu T, Malugin A, Ghandehari H. Impact of silica nanoparticle design    on cellular toxicity and hemolytic activity. ACS Nano. 2011 Jul. 26;    5(7):5717-28.-   Zhang Y, Chen W, Zhang J, Liu J, Chen G, Pope C. in vitro and in    vivo toxicity of CdTe nanoparticles. J Nanosci Nanotechnol. 2007    Feb. 7(2):497-503.

The invention claimed is:
 1. A nanoparticle targeting molecule,comprising: a polymeric network comprising a plurality of first blockcopolymeric segments derived from epoxide monomers and a plurality ofsecond polymeric segments derived from polysaccharides; targeting agentschemically attached to the polymeric network, wherein the targetingagents comprise polyrnyxin B; and a medicament covalently attached tothe polymeric network, wherein the medicament is vancomycin.
 2. Thenanoparticle targeting molecule of claim 1, further comprisingmedicaments encapsulated within the hydrogel.
 3. The nanoparticletargeting molecule of claim 1, wherein the epoxide monomers arepoloxamers.
 4. The nanoparticle targeting molecule of claim 3, whereinthe polysaccharide is dextran.
 5. The nanoparticle targeting molecule ofclaim 1, wherein the nanoparticle targeting molecule has an averagediameter in the range of 1 nanometer to 1000 nanometers.
 6. Thenanoparticle targeting molecule of claim 1, wherein the nanoparticletargeting molecule has an average diameter in a range from 20 nanometersto 250 nanometers.
 7. The nanoparticle targeting molecule of claim 1,wherein the epoxide monomers are poloxamers and the polysaccharide isdextran.
 8. The nanoparticle targeting molecule of claim 1, furthercomprising a biomarker.
 9. The nanoparticle targeting molecule of claim8, wherein the biomarker is a fluorescent biomarker or a radiotagbiomarker.